From synthesis to bio-transformation: control
and management of the quality of APIs
Prof. Marco Terreni
Full Professor in Medicinal Chemistry, University of Pavia; Drug Sciences Department
President of Italian Biocatalysis Center (IBC)
EDQM (Council of Europe, Strasburg): Expert for the Commission of Certification of
Suitability
Qualità di un principio attivo
Nuovi farmaci:
EMEA (ICH Guide line)
Farmaci Generici:
ICH Guide line
Farmacopea
Relevance of the impurity content in APIs
Isolation could be impossible:
levels should be reduced by improving purification processes
Identification by:
GC or HPLC-MS analysis (hypothesis of the possible structure/s)
Chemical synthesis (structure correlation)
General monograph “Substances for
pharmaceutical use” (2034)
• To be applied to “all” substances for pharma use
• Describes the general requirements with regards
quality and purity
• In particular when a specific monograph is not in
compliance with its principles
• Most sections are applicable to fermentation
products and products obtained by bio-processes
General monograph 2034 (cont)
• the Related substances Section does not apply to
fermentation products, but gives principles to be
applied, namely:
– Qualify and specify main impurities
– As much as possible identify impurities and
specify them
– Set a limit of « other impurities »
General monograph (2034)
Peptides
• Generally used at low daily dose
• Many potential related substances: isomers, results
of failures in sequences, cleavages or coupling
• Requirements for related substances included in
the general monograph 2034 from 1/07/2009:
Qualification of Impurities
The level of any impurity present in a new drug substance that has been
adequately tested in safety and/or clinical studies would be considered qualified.
Impurities in known active ingredient can be qualified comparing the impurity
profile with existing commercial products
New impurity in known active ingredient (as reported in Eur. Ph) should be
adequately tested in safety and/or clinical studies for qualification.
• Genotoxicity studies (point mutation, chromosomal aberration)
• General toxicity studies (one species, usually 14 to 90 days)
• Other specific toxicity endpoints, as appropriate
Time consuming
Delayed registration and commercialization of the product
PROBLEMS OF
THE CHEMICAL SYNTHESIS
•Use of toxic solvents
and reagents
•Time-consuming
multi-step procedures
•Formation of by-products
•residual solvents
polluting waste
and
sometimes
low yields
Use of enzymes in the synthesis of active ingredients
i.
ii.
iii.
iv.
v.
vi.
Reaction in aqueous medium (low environmental impact);
Use of no toxic and no pollutants reagents;
Soft reaction conditions;
Avoiding of protection and deprotection steps;
Reduction of the purification steps;
High purity of the final product.
•Lipases
•Esterases
•Amidases
•Glycosidases
•Reductases
•Oxidases
•Transferases
MICRO-ORGANISM
Single step reaction
ISOLATION OF THE ENZYME
Purification
Solid catalyst
Crude extract
Fermentation broth
ENZYME PREPARATION
Free soluble enzymes
Insoluble preparations of pure or almost pure enzymes
Enzymes immobilized on solid supports
Isolated Enzymes
•Fermentation Broth
•Crude extract
Mixture of several proteins
Fermentation-like
•Residuals arising from micro-organism
• Complex downstream
•Purified Enzymes
•Solid or Immobilised Enzymes
Potential Residual Proteins
No use of animal origins raw material in the production of the enzyme
FREE SOLUBLE ENZYMES
S
P
P
S
S
P
S
S
S
P
P
P
z SOLUBLE ENZYMES CAN BE RE-USED
z ALL INACTIVATION MECHANISMS
S
ARE POSSIBLE:
P
S
P
P
REACTORS WITH ULTRAFILTRATION
aggregation
proteolysis
interaction with interfaces
FREE ENZYME ARE OFTEN COMPLEX MIXTURES
SDS-PAGE RML
94,000
67,000
43,000
30,000
20,100
14,400
1
2
3
4
5
6
•Contamination by different proteins
•Side reactions catalyzed by different enzymes
•Other contaminants contained in the crude extract
OPTIMIZATION OF THE BIO-CATALYST:
SOLID AND INSOLUBLE ENZYMES
OPTIMIZED ENZYMES
Re-use of the biocatalyst
Stabilisation of the biocatalyst
Simplification of the downstream
Improved stability
Improved yields
No contamination
ENZYME DERIVATIVES WITHOUT SUPPORT
¾All the solid is enzyme
¾No cost of the support
¾mechanical stability
¾Rigidificaction of the enzyme ?
¾Micro-environment ?
Stabilization of Multimeric Enzymes
High pH
High temperature
Problem
Dissociation of the subunits
in drastic reaction conditions
POSSIBILITIES
Crosslinked Enzyme Crystals
Crosslinked Enzyme Aggregates
CLECs
CLEAs
Pure crystalline enzyme:
Crude enzyme:
requirement of extreme
pure enzyme
Co-aggregation of enzyms
Stabilization of
Multimeric enzymes
Aggregation of enzyme and polymer
Stabilization of multimeric enzymes
Preparation of CLEAs
+
Floculating agent:
PEG
Salts, solvents
CLEAs
Cao L. et al; Org. Lett (2000) 2: 1361-64
cross linking agent
IMMOBILIZATION OF ENZYMES ON
PREEXISTING SUPPORTS
zcovalent multi-point attachment
zreversible immobilization by adsorption
‹ cost of the support
‹ residues after inactivation of the catalyst (covalent
derivatives)
‹ possible release of protein in the reaction medium
Release of protein from immobilised enzymes
Extreme temperature
and presence of co-solvents
HYDROPHOBIC
ADSORPTION
-OOC
Extreme temperature
and pH
No release;
Special case:
multimeric enzymes
IONIC
ADSORPTION
+
+
-
OOC
CH2 HN
COVALENT
ATTACHMENT
CH2 HN
Optimisation of the catalyst
Design of the
enzyme derivative
•Stability
•Catalytic properties
Avoid the Release of protein
in the reaction medium
IMMOBILIZATION BY ADSORPTION
z Easy protocols of immobilization
z The support can be re-used
after enzyme inactivation
Reduction of residues
z Possible product contamination
with the enzyme
Covalent Immobilisation on Epoxidic Supports
O
Enzyme
H2N
pH = 8
OH
H
N
Enzyme
No residual protein
Special case: Multimeric enzymes
Covalent immobilisation
Dissociation of the subunits:
•Low stability of the enzyme
•Residual protein in the final product
Enzymatic synthesis of Ampicillin
NH2
H2N
S
COOCH3
N
O
COOH
Acylase from A. turbidans
40 % of methanol;
Phosphate buffer at
low concentration;
NH2
H
N
S
N
O
O
COOH
Enz. Microb. Technol. 25 (1999) 336-343
Dissociation of the subunits:
Low stability of the enzyme
In the synthetic conditions
AN INTEGRATED APPROACH TO THE
STABILIZATION OF MULTIMERIC ENZYMES
1. MULTI - SUBUNIT COVALENT IMMOBILIZATION
2. SUBUNIT CROSSLINKING WHIT POLYFUNCTIONAL MOLECULES
CHO
CHO
CHO
CHO CHO
CHO
Polyaldehydes
Stability of the enzyme derivatives
Residual activity, (%)
100
Crosslinked derivative
75
50
25
Free enzyme
0
0
10
20
TIME, (hours)
30
40
Immobilised Enzyme
Control of residual proteins
Knowledge of the catalyst engineering
Suitability of the downstream process
Test for release of proteins in the
reaction condition
Test for residual proteins in the
final product
1. Fermentation Processes
General monograph « Products of
fermentation » (1468)
• Scope:
– « Indirect » gene products obtained by fermentation
• Out of the scope:
– Vaccines, products from continuous cell lines, of
animal/human origin
– Direct gene products
– Semi-synthetic products, biocatalytic process
• Provides general requirements for manufacture of
fermentation products. Compliance mandatory for these
products
FERMENTATION
Advantages
•One step reaction: synthesis of complex molecules
• Low costs
Disadvantages
•Complex mixtures
•Contaminants with different structures
•Residuals arising from micro-organisms
• Complex downstream
•Complex separation and analytical procedure for control of
impurities
Product of fermentation Complex structures
Simple compounds, normally prepared by chemical
synthesis, are more and more prepared by
fermentation.
For example:
•Aminoacids
•Nucleosides and nucleotides
•Vitamins
Control of the quality is depending from the process used.
Requirements
• Source and history of the producer micro-organism
• Characterisation, stability, of the producer microorganism
• Detailed description of fermentation, incl materials,
preparation of media, downstream processing
• In-process controls at all stages
• Purity of the final substance: impurity profile and
specification
Impurity profile
• Impurities (3.2.S.3)
– Describe all potential related substances
(sometimes complex), focus on actual impurities
– Address residual solvents according to European
guidelines
– Discuss particular impurities arising from
fermentation: residues of substrates, cells
residues, proteins,…
Limits for related substances
• Set limits according to the monograph
• Set limits for the other known impurities
– Qualify impurity profile by comparison with
products already on the market, or tox data
– In line with levels found in batches
• Set limits for unknown impurities + total
impurities
Limits for residual solvents
• According to ICH and CHMP guidelines on
Residual solvents
• Show absence of the solvents used during
purification
• Set limits and propose methods for
solvents used during purification
Other impurities
• Demonstrate that there are no residues from
fermentation
• Proteins: not a concern for oral use (a lime
proposed), to be addressed if parenteral use
(absence should be demonstrated)
• DNA
• The Ph. Eur provides general methods which
can be used
downstream:
Elimination of residuals from micro-organism
•Extraction with organic solvents
allows complete elimination of residuals. Presence of residual indicate a not
well separation of phases.
•Crystallization in organic solvent
Most adequate for residuals elimination
•Ultra filtration
Residuals of low molecular weight proteins and peptides
•Chromatographic columns
Complete elimination of protein is ensured depending from the
chromatographic conditions
Problems for high water soluble products:
•Peptides
•Amino acids
•Oligosaccharides
•Nucleosides and nucleotides
Evaluation of residuals arising from the micro-organism
•Absence of residuals from the microorganism should be demonstrated
during process validation
•Product for injection should be carefully controlled
Nucleic acids
•Absorbance tests
•RT-PCR or PCR
Proteins
•Colorimetric tests: Bradford, Lowry or other according to the
Monograph for Assay of Total Protein 2.5.33 (01/2008:20533)
•Electrophoresis
Example of fermentation product:
L-serine
Isolation from fermentation broth:
• Filtration of biomass
• Anionic and cationic exchange resins
Purification:
• Ultrafiltration of water solution (cut-off m.w.>6000)
• Crystallization from water
Possible impurities:
Aminoacids. Analysis performed with A.A. analyzer (each NMT 0.2%;
total NMT 1.0%).
Absence of protein demonstrated by Bradfford assay (colorimetric: LOD
1ug/mL)
Example of Enzymatic bioprocess: L-serine
Glicine
Enzyme
L-Serine
Enzyme Isolation from fermentation broth:
• Filtration of biomass
• Solution is directly used without purification of the enzyme
Purification:
• Ionic exchange colums
• Crystallization from organic solvent/water solution
• Ultrafiltration of water solution (cut-off m.w.>5000)
• Crystallization from water
Possible impurities:
Glicine (starting material) and other A.A. Analysis performed with A.A. analyzer (each
NMT 0.5%; total NMT 1.0%).
Absence of protein demonstrated by adsorbance at 650 nm (LOD 40 ppm)
Example of fermentation product:
Teicoplanine….a long story
Is a Complex mixture of products. Problems related to:.
•Identification
•Quality
Teicoplanine
Composition of the “Complex”
and related substances are strictly dependent from:
•Microorganism
•Control of the Fermentation process
•Raw material used for fermentation process
Teicoplanine New Producer
Japan Pharcopoea (JP)
JP
Specification
TA2 Group
NLT 80%
TA3 Group
NMT 15%
Other
NMT 5%
Teico
Batch A
95.24
Teico
Batch B
94.04
Teico
Batch C
90.50
3.71
4.45
8.28
1.05
1.51
1.22
Commercial Medicinal product (Tangosit) and the new Teicoplanine API
meet specification for JP
Csilla FranK; Controllo della qualità di un prodotto di fermentazione: aspetti tecnici e regolatori
Thesis “Master in Discipline Regolatorie 2008”; University of Pavia; Pavia; Italy
Teicoplanine: satisfaction of EP Specification
Conform with the Specification
Out of Specification
EP
Specification
Teico
Batch A
Teico
Batch B
Teico
Batch C
TA2-1 Group
NMT 20%
10.65
6.32
8.62
TA2-2 Group
NMT 35-55%
59.54
56.54
TA2-3 Group
NMT 20%
9.04
TA2-4 Group
NMT 20%
TA2-5 Group
NMT 20%
EP
Specification
Teico
Batch A
Teico
Batch B
Teico
Batch C
TA2-1 Group
NMT 20%
9.3
8.3
7.7
55.01
TA2-2 Group
NMT 35-55%
47.7
47.7
48.7
10.69
8.95
TA2-3 Group
NMT 20%
8.2
7.5
7.0
4.27
7.51
5.05
TA2-4 Group
NMT 20%
14.8
14.1
13.5
2.79
5.26
3.57
TA2-5 Group
NMT 20%
6.7
7.1
7.1
Modulation of fermentation condition
Selection of the ingredient used during fermentation
Csilla FranK; Controllo della qualità di un prodotto di fermentazione: aspetti tecnici e regolatori
Thesis “Master in Discipline Regolatorie 2008”; University of Pavia; Pavia; Italy
Limit for impurities in Teicoplanin?
According to Guide line on setting specification for related
impurities in antibiotics
Proposed limits for Active Substances manufactured by
fermentation: Fanmily of compound
Reporting threshold:
0.10%
Identification threshold
0.15%
Qualification threshold unknown
0.15%
Qualification threshold related
Identification at least by HPLC-MS
0.50%
Any other peak in the chromatogram should be identified and
confirmed to have a teicoplanin-like structure.
EVALUATION OF TEICOPLANINE QUALITY
1. Evaluationof the complex according to the EMA guide line.
2, Compariosion of the new product with EP-CRS and commercial product
3. Confirmation of Teicoplanine-like impurities by HPLC-MS analysis
4. Specification for impurities >0.5% specified by RRT
5. Revision of the EP monograph under evaluation accordingly